1. Field of the Invention
The invention relates to fusion proteins containing sequences from the complement inhibitors membrane co-factor protein (hereinafter `MCP`) and decay accelerating factor (hereinafter `DAF`) wherein the fusion proteins also contain at least one cell surface localizing domain.
2. Description of the Prior Art
Complement is an important defense mechanism for warding off infectious agents. Complement works by targeting microorganisms and other antigens to complement-receptor-bearing cells, recruiting phagocytic cells to the area where complement activation is taking place and the destruction of target membrane.
Complement is a cascade of over 30 proteins and is turned on during inflammation. Indeed, the complement components C3a, C4a and C5a trigger the release of inflammatory mediators from mast cells, induce smooth muscle contraction, increase vessel permeability and recruit neutrophils. In some cases, inflammation and the accompanying activation of complement results in damage to host tissues. Sepsis, Adult Respiratory Distress Syndrome (hereinafter `ARDS`), reperfusion injury and burns are among the clinically significant conditions in which amplification of complement activation results in tissue damage.
Complement is regulated in part by six proteins with closely related primary sequence structure including Membrane Cofactor Protein (hereinafter `MCP`) and Decay Accelerating Factor (hereinafter `DAF`). The genes encoding MCP and DAF, as well as four other complement regulating proteins have been localized to the long arm of human chromosome 1, band 1q32. All six of these proteins have a common structural motif with an approximately 60-amino acid consensus unit or short consensus repeat (hereinafter `SCR`) and are present in four contiguous copies in MCP and DAF. Although not completely identical in sequence, the SCRs have invariant cysteines at four positions and up to 18 highly conserved positions throughout the rest of the sequence.
MCP (also known as `CD46`) is present on the cell surface of a number of cell types including peripheral blood cells (excluding erythrocytes), cells of epithelial, endothelial and fibroblast lineages, trophoblasts and sperm. MCP has four SCR sequences and serine/threonine enriched region in which heavy O-linked glycosylation occurs. MCP also has a transmembrane and cytoplasmic domain. MCP works by binding to the C3b and C4b present on the cell surface thereby targeting the protein for degradation by factor I, a plasma protease, and thereby destroying any C3 or C4 convertase activity.
Thus, MCP is said to have "cofactor activity". Because MCP is localized on the cell surface, it protects only the cells on which it is present and is therefore said to act in an intrinsic manner. The sequence of a cDNA encoding human MCP has been reported by Lublin et al, J. Exp. Med., (1988) 168:181-194.
DAF is present on the cell surface of a number of cell types including peripheral blood cells (including erythrocytes), cells of epithelial, endothelial and fibroblast lineages, trophoblasts and sperm. DAF regulates complement function via "decay accelerating activity". That is, DAF binds to C4b/C2a and to C3b/Bb and destabilizes the association of C2a or Bb (the protease component) thus destroying the C3 convertase activity. DAF has also been reported to interfere with the formation of C4b/C2a and C3b/Bb complexes. As with MCP, DAF regulates complement in an intrinsic manner, thus protecting only the cells on which DAF is located.
The sequence of cDNA encoding human DAF has been reported by Medof et al, Proc. Nat. Acad. Sci. USA (1987) 84:2007, and by Caras et al, Nature, (1987) 325:545-549. The disclosure of both articles is herein incorporated by reference. Like MCP, DAF has four SCR sequences and a serine/threonine enriched region in which heavy O-linked glycosylation occurs. DAF has a cell surface localizing domain at it carboxy terminal end at which a glycophosphatidylinositol moiety (hereinafter `GPI`) is covalently bound. The GPI links DAF to cell surfaces and even allows for reattachment of DAF to cell surfaces after solubilization of cell membranes with detergent. Caras et al report the finding of two mRNAs encoding DAF. One species encodes DAF with the cell surface localizing domain. The second species accounts for 10% of DAF mRNA and appears to encode a secreted species of DAF which is lacking the cell surface localizing domain to which the GPI-anchor is attached.
This cell surface localizing domain appears to be important for the optimal function of DAF. Patients with paroxysmal nocturnal hemoglobinuria are known to be deficient in GPI-anchored proteins and this deficiency is known to causally related to susceptibility of their blood cells to lysis by complement. Moreover, Moran et al, J. Immunol,. (1992) 149:1736-1743 have shown that recombinant full length DAF and recombinant DAF which is missing the cell surface localizing domain (seDAF) both protect cells against complement, but that mDAF was 50 fold more potent than seDAF. However, mDAF must be incorporated into cell surface membranes to have the higher level of activity and it appears that serum lipoproteins interfere with this incorporation. This observation led Moran et al to the conclusion that seDAF will be the preferred molecule for clinical applications.
Lublin and Coyne, J. Exp. Med. (1991) 174:35-44 compared the activity of DAF, MCP, and variants of DAF and MCP in which the cell-surface membrane localizing domains of DAF and MCP (the GPI-anchor of DAF and the transmembrane (TM) domain of MCP) had been exchanged. Interestingly, DAF and the DAF/MCP-TM variant showed approximately equal complement inhibiting activity. Likewise, MCP and the MCP/DAF-GPI anchor variant showed approximately equal complement inhibiting activity in vitro.
Hybrid complement regulatory proteins containing DAF and MCP in a single polypeptide have been disclosed. See eg. Iwata et al, J. Immunol., (1994) 152:3436-3444. Iwata et al disclose that MCP-DAF hybrids are more effective than DAF, MCP or DAF and MCP at inhibiting C3 deposition via the alternative pathway. The MCP-DAF hybrid was also more effective than MCP alone at inhibiting C3 deposition via the classical pathway.